Random famplified polymorphic DNA, PCR, RAPD

1. Random amplified Polymorphic DNA
(RAPD)
DR RADHAKRISHNA G PILLAI
Department of Life Sciences
University of Calicut
Kerala, INDIA 673 635

2. RAPD
• RAPD (pronounced "rapid") stands for 'Random
Amplified Polymorphic DNA‘
• It is a type of PCR reaction, but the segments of DNA
that are amplified are random
• Several arbitrary, short primers (8–12 nucleotides)
are used
• PCR using a large template of genomic DNA
• Fragments may amplify
• A Pattern OF PCR product obtained
• A semi-unique profile can be obtained from a RAPD
process

3. Uniqueness of RAPD
• No knowledge of the DNA sequence for the
targeted genome is needed as the primers bind
random
• This makes the method popular for comparing
the DNA of biological systems that are not well
studied
• Because it relies on a large, intact DNA template
sequence

4. RAPD
• No fragment is produced if primers annealed too far
apart or 3' ends of the primers are not facing each
other
• If a mutation has occurred in the template DNA at the
site that was previously complementary to the primer,
a PCR product will not be produced, resulting in a
different pattern of amplified DNA segments on the
gel
• Selecting the right sequence for the primer is very
important
• Different sequences will produce different band
patterns
• More specific recognition of individual strains by
different primers

5. Limitations of RAPD
• Resolving power is much lower than targeted, species
specific DNA comparison methods, such as short tandem
repeats.
• Nearly all RAPD markers are dominant, i.e. it is not possible
to distinguish whether a DNA segment is amplified from a
locus that is heterozygous (1 copy) or homozygous (2 copies)
• Codominant RAPD markers, observed as different-sized DNA
segments amplified from the same locus, are detected only
rarely.
• RAPD technique is laboratory dependent and needs
carefully developed laboratory protocols to be reproducible.
• Mismatches between the primer and the template may
result in the total absence of PCR product as well as in a
merely decreased amount of the product. Thus, the RAPD
results can be difficult to interpret

7. RAPD
• 100 to 3000 bp depending upon the genomic
DNA and the primer
• This technique is sensitive, fast, requires the
use of no radioactive probes, and is easily
performed.
• Computer programs available to analyse
RAPD profile

8. Amplified Fragment Length Polymorphism
(AFLP)
• AFLP is A PCR based tool used in genetics research, DNA
fingerprinting, and in genetic engineering
• AFLP uses restriction enzymes to digest genomic DNA
• Ligation of adaptors to the sticky ends of the restriction
fragments
• A subset of the restriction fragments is then selected to be
amplified
• This selection is achieved by using primers complementary to the
adaptor sequence, the restriction site sequence and a few
nucleotides inside the restriction site fragments
• The amplified fragments are separated and visualized on
denaturing PAGE
• Autoradiography or fluorescence methodologies used for
identification
• Sequencing could also be used for identification

9. AFLP
• The AFLP technology has the capability to detect
various polymorphisms in different genomic
regions simultaneously
• Highly sensitive and reproducible
• ALP is widely used for the identification of genetic
variation in strains or closely related species of
plants, fungi, animals, and bacteria
• Also used in criminal and paternity tests
• To determine slight differences within populations
• Linkage studies to generate maps for quantitative
trait locus (QTL) analysis.

10. Advantages of AFLP
• AFLP has higher reproducibility, resolution, and
sensitivity at the whole genome level compared to
other techniques –RAPD, RFLP AND microsatelite
• Has the capability to amplify between 50 and 100
fragments at one time
• No prior sequence information is needed for
amplification
• AFLP is extremely beneficial in the study of taxa
including bacteria, fungi, and plants, where much is
still unknown about the genomic makeup of various
organisms.